This project is to study gene mutations and small intrachromosomal changes directly in exposed mammals. We have taken two approaches. 1) Site specific alterations in proteins detected in single cells: Antigenic differences between rat and mouse LDH-C were used to detect mutations in mouse sperm. Initial work indicated that this system had potential for the study of mutagenesis in mammals; later research showed that the apparent mutation frequency varied too much to give reproducibly useful data. The conclusion was reached that although antibodies had the needed sensitivity, interspecies differences may not be appropriate as markers for induced mutations; also, a model mutant cell must exist for a proper test of antibodies. Therefore, use of antibodies are being applied to spontaneous and induced mutants of malic enzyme (MOD-1) in mice. Normal (MOD-1) was purified and antibodies produced; sperm and somatic cells were labeled with this antibody. A mutant form of MOD-1 was purified. The future strategy is to produce and test antibody to the abnormal MOD-1 using cells from normal homozygotes and heterzygotes with the mutant allele. 2) Site specific changes detected at the DNA level: A major problem for detection of genetic damage directly in mammalian DNA is that most genes occur in one, or few copies; there are, however, two possibilities. The first is the use of mitochondrial (mt) DNA. Cloned mouse mtDNA has been used for restriction analysis of sperm mtDNA isolated from a single mouse; after additional technical improvements, the mtDNA from treated mice will be examined for mutations. The second is the use of viral DNA transformed into mammalian DNA. Double stranded DNA from PhiX174 am3, cs70 was transformed into mouse L-cells. The DNA was incorporated into several places in tandem arrangements. Using restriction enzymes and ligase it was possible to transfect spheroplasts with PhiXDNA from the transformed mammalian cells. Attempts are being made to create a mouse strain with PhiXDNA in the genome for the study of mutation induction in any part of the animal tissue.